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Genetics of pathogenicity in Pyrenophora leaf diseases of barley

Campbell, Graham F. (Graham Findlay) (2001-12)

Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001.

Thesis

ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most
important diseases of this cereal in the south Western Cape Province of
South Africa. This fungus exists as two different types (forms), namely a nettype
and a spot-type that are distinguished by differential symptom expression
on barley leaves. Based on this specific plant pathological difference a series
of studies of agricultural importance were executed to investigate the effects
of sexual recombination between these two types. In addition, studies were
done to determine the difference between local net- and spot-type populations
with regards to population structure and fungicide sensitivity. This dissertation
therefore, consists of a collection of separate publications and as a result a
certain degree of redundancy has been unavoidable.
Recombination is one of the most important evolutionary forces
involved with sexual reproduction. In plant-fungal agricultural ecosystems this
may result in pathogenic fungal populations adapting more rapidly to control
programs such as fungicide applications. The first section of the review in
part 1 of this dissertation covers different aspects of sexual reproduction in
ascomycetes, specifically focussing on mating-type genes, vegetative
incompatibility and recombination. The major part of the review is then
dedicated to various plant pathological aspects of P.teres, specifically
addressing the differences between the two types, and in various cases
highlighting the significance of sexual recombination within and between the
net- and spot-type.
Using morphological criteria for identification purposes there have been
many conflicting reports concerning the identity of leaf spot isolates in the
Western Cape Province of South Africa. In part 2, the correct identity was
eventually achieved employing mating studies and molecular markers .: This
was accomplished after single ascospores were obtained from pseudothecia
after in vitro mating had occurred between a verified P. teres net-blotch isolate
from Denmark and a representative Pyrenophora leaf spot isolate from South
Africa. Using amplified fragment length polymorphism (AFLP) and RAPD
markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials
also confirmed that recombination had taken place during mating.
Inoculations were conducted on the differential cultivars susceptible to the
net-blotch and leaf spot forms. The two parents induced typical net-blotch or
leaf spot symptoms whereas the progeny mostly induced a jagged spot
symptom on each cultivar. Fungicide sensitivity tests using the ergosterol
biosynthesis inhibitors showed that, due to recombination, some progeny
could have increased resistance to these fungicides. Due to mating and
subsequent recombination between a net blotch isolate of P. teres and a
representative leaf spot isolate, it was concluded that the latter was P. teres f.
maculata.
Fifteen of the net-spot hybrid progeny (F1) produced from the mating
study in Part 2 were screened in Part 3 to assess their viability and genetic
stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of
the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and
Clipper (both differentially susceptible to spot-type isolates) produced
intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from
foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a
healthy set of seedlings produced similar intermediate symptoms. RAPDs
conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny
revealed profiles similar to those obtained for F1 isolates. RAPD molecular
data, therefore, indicated that hybrid progeny of this net x spot mating were
genetically stable after having been subjected to two repetitive inoculation and
reisolation cycles. Phylogenetic analysis of DNA sequences of the internal
transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA
gene and the 5' end partial histone-3 gene confirmed the genetic stability of
the hybrid progeny. These results also indicated that the hybrid progeny
produced consistent symptoms throughout the series of experiments, and
maintained their virulence to the differential cultivars screened.
Both types of P. teres are prevalent in the south Western Cape
Province of South Africa, found on susceptible cultivars often grown within
close proximity of each other. In Part 4, a net- and spot-type population were
characterised in terms of their population structure using RAPD markers.
Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields,
diagonal to one another. A total of 65 loci were produced of which 54 were
polymorphic. Total gene diversities determined for all loci resulted in mean
indices of 0.063 and 0.082 being obtained respectively for the net- and spottype
populations. A coefficient of genetic differentiation (Gs) of 0.0149 was
obtained between sites within populations while a coefficient (GT) of 0.63 was
obtained between the two populations. Genotypic variation revealed 13
distinct multilocus genotypes (haplotypes) in the net-type population while
there were 12 in the spot-type population. UPGMA cluster analysis done on
the two populations together with six progeny from the mating between a netand
spot-type isolate resulted in three main clusters being produced, one for
each population and one for the progeny. One isolate collected from the nettype
population also contained a unique spot-type RAPD fragment. This
suggested that sexual recombination may be taking place between isolates of
the net- and spot-type under field conditions.
Fungicide application is the most important method used in the control
of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values)
of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol
demethylation inhibiting fungicides were determined, based on the inhibitory
effect on radial mycelial growth. The fungicides evaluated were triadimenol,
bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and
spot-type isolates revealed strong resistance to triadimenol while flusilazole
was shown to be the strongest inhibitor of fungal growth. Spot-type isolates
showed a higher resistance than net-type isolates to all five fungicides
screened. The ICsD values indicated significant differences between four of the
fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The
ICsD values between propiconazole and bromuconazole were not significant.
This study suggested that spot-type isolates showed a higher degree of
resistance to commercially used fungicides than net-type isolates.
The overall conclusion of this study is that the spot-type of P. teres is
the pathogen associated with leaf spots of barley in the south western Cape
province of South Africa and not P. japonica as earlier reported. Together
with the net-type, both types exist as genetically variable populations in this
barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to
one another in which either net- or spot-type susceptible cultivars are being
cultivated may lead to sexual progeny being produced. This in turn may lead
to an increased rate at which fungal populations may become resistant to
commercially used fungicides. It is furthermore suggested that an alternative
fungicide seed treatment is used instead of triadimenol due to high resistance
of P. teres to this fungicide.